The application of gene therapy for the treatment of human disease has increased steadily since the first human gene therapy trial was conducted in 1989. To date, more than one hundred gene therapy protocols and clinical trials have been approved by the Recombinant DNA Advisory Committee (RAC) for the treatment of inherited and acquired diseases. Despite the reported advances in gene therapy technology and the increase of approvals in gene therapy protocols, obstacles still remain, including the difficulty in efficient delivery of exogenous genes in vivo.
Gene therapy generally involves the introduction and expression in an animal of an exogenous gene to supplement or replace a defective or missing gene or to produce a product for treating an acquired disease. While there remains some debate about which vectors are most useful under which circumstances, the evolving challenge is not whether gene therapy will work, but rather determining which vectors are the most effective and which delivery schemes are most effective for carrying out gene therapy.
Among the difficulties with delivering exogenous genes to cells for gene therapy is the cell wall itself Some vectors and naked DNA do not efficiently penetrate mammalian cell walls. This is less of a problem in ex vivo applications where a variety of physio/chemical and mechanical technologies have been developed for introducing genes into cells. Many of these techniques, however, cannot be applied in vivo. Another problem with in vivo delivery of genes to cells is that complex structures such as vectors containing genes under the control of promoters do not fare well in certain physiological environments and are destroyed. These larger complex DNA molecules are unlike short antisense oligonucleotides which typically are modified to protect them against physiological degradation.
The conditions that can destroy an operable gene in vivo are not the only barriers to delivery of genes for gene therapy. The preparative techniques for formulating delivery systems can be destructive to DNA as well. For example, many procedures for forming microparticles require high temperature and/or high sheer forces and/or sonication. Such conditions typically""would destroy a vector containing a gene or would result in breakage of a large piece of DNA.
Oral formulations of drugs, although most convenient to the patient, face severe obstacles to delivering the drug molecules to the target cells. This is particularly true for labile drugs such as pieces of DNA or genes. A first obstacle is the stomach. The environment of the stomach is extremely destructive to DNA, and most DNA (and especially large pieces of DNA) would not survive the environment of the stomach. Even if the DNA did survive the environment of the stomach, it then must be taken up by or passed between the cells lining the large and small intestine. Uptake of material across a mucosai epithelial barrier is a selective event, and not all molecules would be expected to be taken up by absorptive and nonadsorptive epithelial cells and/or taken up into systemic circulation. Even if this obstacle is overcome, then the DNA still must resist destruction when in general circulation. The DNA also must gain access accross the membrane of the target cell which is to be transplanted. Finally, the DNA must be presented in a fashion that is nontoxic to the subject. For example, some viral vectors have been shown to induce severe immunological responses in the recipients and some liposomes have been shown to be toxic to recipients.
U.S. Pat. No. 5,075,109, entitled xe2x80x9cMETHOD OF POTENTIATING AN IMMUNE RESPONSExe2x80x9d, issued to Tice, is directed to methods for oral administration of a bioactive agent contained in microparticles to protect the agent from degradation during its passage through the gastrointestinal tract. The patent is particularly directed to a method of oral immunization which will effectively stimulate the mucosal immune system and overcome the problem of degradation of the bioactive ingredient during its passage through the gastrointestinal tract to the Peyer""s patch. The ""109 patent involves administering bioactive agents contained in microcapsules that are sized between approximately one and ten microns. The microcapsules apparently survive the environment of the stomach and are taken up by the Peyer""s patches to stimulate the immune response. The ""109 patent does include the term xe2x80x9cnucleic acidsxe2x80x9d as a member of a long list of materials regarded as xe2x80x9cbioactive agentsxe2x80x9d. The ""109 patent does not mention the delivery of genes, the delivery of genes under the control of a promoter or the delivery of vectors including genes. This is perhaps because the methodology employed by Tice in making the microparticles is typical of prior art fabrication techniques, that is, aggressive emulsification conditions are applied, such as would destroy large pieces of DNA, in order to form the microparticles.
In none of the prior art of which applicants are aware is there disclosed the notion of delivery in microparticles of genes under the control of promoters. Certainly, none disclose the notion of oral delivery of genes under the control of promoters.
It is an object of the invention to provide a noninvasive method of carrying out gene therapy.
Another object of the invention is to provide an oral means of carrying out gene therapy.
Another object of the invention is to provide a method for microencapsulating large pieces of DNA, such as genes under the control of promoters and vectors, in a manner which does not destroy the DNA and that produces a high yield of DNA within the microcapsule. These and other objects are achieved by the present invention.
The invention involves the discovery of a method for encapsulating oligonucleotides in a nondestructive fashion and in high yield. The invention further involves the discovery that microparticles can be used to deliver these oligonucleotides orally and in functional form, not only to intestinal epithelial cells but also to nonepithelial cells within the gastrointestinal system (e.g. Peyer""s patches) and even to cells remote from the intestinal epithelium such as spleen or liver cells. The invention further involves the discovery that bioadhesive microspheres, instead of simply increasing residence time upon attachment to a mucosal epithelium, surprisingly, are: (1) taken up into the epithelial cells, including absorptive intestinal epithelial cells; (2) taken up into gut associated lymphoid tissue; and (3) even transported to cells remote from the mucosal epithelium. The microparticles containing the oligonucleotides preferably are between 10 nanometers and five microns. In some important embodiments, the microparticles have an average particle size consisting of between 100 nanometers and three microns. Most preferably, the microparticles are prepared by phase inversion nanoencapsulation. The oligonucleotides are in bioactive form when released from the microparticles.
Surprisingly, we have established that genes under the control of promoters can be protectively contained in microparticles and delivered to cells in operative form, thereby obtaining noninvasive gene delivery for gene therapy. The invention overcomes extraordinary obstacles: (1) the genes are not destroyed, disrupted or inactivated by the manufacturing technique for producing the microparticles; (2) the microparticles protect the genes from the destructive environment of the stomach; (3) the microparticles enter the target cells; (4) the microparticles cause transfection of the cells with the genes; (5) the microparticles can deliver the genes to sites remote from the mucosal epithelium, i.e. can cross the epithelial barrier and enter into general circulation, thereby transfecting cells at other locations.
According to one aspect of the invention, a method for delivering a gene to a cell of a subject for gene therapy is provided. An effective amount of bioadhesive microparticles containing an isolated gene under the control of a promoter is administered noninvasively to a mucosal epithelial surface of a subject in need of such treatment. Preferably the bioadhesive microparticles consist of microparticles having an average particles size of between ten nanometers and five microns. In one embodiment, the microparticles consist of microparticles having an average particle size of between one hundred nanometers and three microns. The preferred bioadhesive microparticles comprise polyanhydrides, most preferably poly(fumaric-co-sebacic)anhydride. Preferably, they also contain metal oxides or hydroxides. Preferably, they further contain anhydride oligomers. Most preferably the bioadhesive microparticles have bioadhesive properties at least as strong as 20:80 poly(furaric-co-sebacic)anhydride.
The microparticles can be administered non-invasively, such as by oral formulation and by aerosols for the respiratory tract. In some embodiments, the gene is delivered to and transforms an epithelial cell. In other embodiments the gene is delivered to and transforms a nonepithelial cell.
According to another aspect of the invention, a method is provided for delivering a gene to a cell of a subject for gene therapy. An effective amount of microparticles containing an isolated gene under the control of a promoter is administered orally to a subject in need of such treatment. The microparticles consist of microparticles having an average particle size of between ten nanometers and five microns. In one embodiment, the microparticles have an average particle size of between one hundred nanometers and three microns.
The microparticles may be delivered to and transfect an epithelial cell or may be delivered across such epithelial cells to nonepithelial cells which are transformed by the gene. In one embodiment the cell is a gut associated lymphatic tissue cell. In another embodiment the cell is an adsorptive epithelial cell. In yet another embodiment, the microparticle is taken up into systemic circulation and the cell transfected is a nonepithelial cell remote from the epithelial barrier, such as, for example, a spleen cell or a liver cell. It is preferred that the microparticles are bioadhesive microparticles, as described above.
According to another aspect of the invention, a method is provided for noninvasive delivery of a gene into systemic circulation of a subject for gene therapy. Microparticles containing a gene under the control of a promoter are administered noninvasively to a mucosal epithelial surface of a subject in need of such treatment. The microparticles are in an effective amount and consist of microparticles having an average particle size of between ten nanometers and five microns. In one embodiment the average particles size is between one hundred nanometers and three microns. The modes of delivery and the preferred microparticles are as described above.
According to another aspect of the invention, an article of manufacture is provided. The article of manufacture is a preparation consisting essentially of microparticles containing an isolated gene under the control of a promoter. The microparticle preferably is bioadhesive. In one embodiment the microparticles consist of microparticles having an average particle size of between ten nanometers and five microns. In other embodiments, the microparticles consist of microparticles having an average particle size of between one hundred nanometers and three microns. The preferred bioadhesive microparticles are as described above.
According to another aspect of the invention, a pharmaceutical preparation for gene therapy is provided. The preparation contains an effective amount of microparticles containing a gene under the control of a promoter, wherein the microparticles consist of microparticles having an average particle size of between ten nanometers and five microns. In some embodiments, the microparticles consist of microparticles having an average particle size of between one hundred nanometers and three microns. The preparation also can include a pharmaceutically acceptable carrier suitable, for example, for oral administration to a subject wherein the pharmaceutical preparation is formulated as an oral dosage.
The invention also involves the use of any one of the foregoing materials for gene therapy. A particularly important embodiment involves a formulation for oral administration or administration by inhalation.
These and other aspects of the invention are described in greater detail below.